Assay method utilizing capillary transport on non-porous substrates

ABSTRACT

A device and method for the separation of a component in a liquid sample prior to the detection of an analyte in said sample, wherein a sample is added to a receiving zone on a substrate, said substrate further optionally comprising a reaction zone, a transport or incubation zone connecting the receiving and reaction zone, respectively, forming a flow path on a substrate, wherein said substrate is a non-porous substrate, and at least part of said flow path consists of areas of projections substantially vertical to said surface, and having a height (H), diameter (D) and reciprocal spacing (t 1 , t 2 ) such, that lateral capillary flow of said liquid sample in said zone is achieved, and where means for separation are provided adjacent to the zone for receiving the sample. Said means for separation are chosen among filter means, optionally enhanced by affinity binding and/or aggregation; magnetic means, also optionally enhanced by affinity binding and/or aggregation; and acoustic means.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. patentapplication Ser. No. 10/560,138, filed Dec. 9, 2005, which is a U.S.National Phase of PCT/SE2005/000429, filed Mar. 23, 2005, which claimspriority to Swedish Patent Application Serial No. SE 0400662-3, filedMar. 24, 2004, all of which are hereby incorporated by reference intheir entirety.

The present invention concerns an assay device or assay system or acomponent thereof, for use in the detection of one or more analytes in asample, as well as method for the use of said device or component, andmethods for detection of an analyte, using said device. The invention inparticular concerns devices, components and methods for applications,where a component of the sample needs to be separated before or duringthe reaction for detection of the analyte.

BACKGROUND

Analytical and diagnostic determinations are frequently performed onliquid samples, comprising in addition to the analyte of interest, alsocountless other components, in solution and/or in particulate form,which often interfere with the handling of the sample and may influencethe quantitative or qualitative determination of the analyte.

For example, numerous clinical diagnostic methods are based on thedetection of an analyte in a biological sample. Frequently, suchdetection is achieved in a disposable assay device, allowing rapid andsimple diagnosis. One important application is the wide field ofimmunology, where analytes are detected with the aid of specificantibodies, capable of binding to the analytes and forming detectablecomplexes, usually with the aid of ligands aiding the detection.

When performing a test using a biological sample from a patient, inparticular a blood sample, many factors need to be considered. Wholeblood is prone to clotting, reducing or preventing the desired flow ofthe sample in the assay device. The red blood cells, even in the absenceof clotting, may inhibit or retard flow. Further, red blood cells mayinhibit binding between specific binding pair members. Red blood cellsalso have enzymatic activity, which, depending on the assay employed,may interfere with the signal produced.

Unfortunately, red blood cells present in whole blood also scatter andabsorb light thus interfering with assay methodologies which measureeither reflected or transmitted light. Also other cells may interferewith particular determinations; for example, cholesterol determinationscan be effected by cholesterol present in cell membranes.

Further, the red cell fraction takes up a considerable volume of thesample, in some cases as much as half the volume. Importantly, thisfraction, also called hematocrit, may vary between different individualsand even in the same individual, between different measurements. This inturn may influence the accuracy and/or the repeatability of thedeterminations.

Consequently many assays involve a step of separating the red bloodcells, whereupon the assay is carried out on plasma or serum. When theseparation is performed before clotting, plasma is obtained. Whenclotting has occurred before separation, serum is obtained.

The red blood cells can be separated from plasma through centrifugation,which however requires a relatively large volume of sample, and the useof a centrifuge. This is also time consuming and constitutes anadditional step of handling the sample, which increases cost andcomplexity, and which further should be avoided in particular whenpotentially contagious blood-borne pathogens are involved. Additionally,the risk of the sample being contaminated by the individuals handlingit, cross-contaminated by parallel samples or mixed up with othersamples is increased.

What is said above regarding whole blood samples and red blood cellsapplies also, with necessary adaptations, to all other biologicalsamples, where cells, cell debris, fibres, or other unwanted particlesetc., may interfere with the determination and should thereforepreferably be separated before or during the reaction or determinationleading to the detection of the analyte.

The most common type of disposable assay device consists of a zone orarea for receiving the sample, a reaction zone, and optionally atransport or incubation zone connecting the receiving and reaction zone,respectively. These assay devices are known as chromatography assaydevices or simply referred to as strip tests. They employ a porousmaterial defining a path for fluid flow capable of supporting capillaryflow, e.g. a filter material. The sample-receiving zone frequentlyconsists of a more porous material, capable of absorbing the sample,and, when the separation of blood cells is desired, effective to trapthe red blood cells. Examples of such materials are fibrous materials,such as paper, fleece, gel or tissue, comprised e.g. of cellulose, wool,glass fibre, asbestos, synthetic fibres, polymers, etc. or mixtures ofthe same. The transport or incubation zone commonly consists of the sameor similar materials, often with another porosity than thesample-receiving zone. Likewise, the reaction zone, which may beintegrated with the incubation zone, or constituting the most distalpart thereof, commonly consists of similar, absorbing fibrous materials,or any of the above listed materials.

In an assay device or strip test, the porous material(-s) is (are)assembled on a carrier, such as a strip of thermoplastic material,paper, cardboard or the like. Further, a cover can be provided, saidcover having at least one aperture for receiving the sample, and anaperture or transparent area for reading the result of the assay.

Nitrocellulose materials are also frequently used as the matrixconstituting the transport or reaction zone, connecting the receivingzone and the reaction zone. A significant disadvantage withnitrocellulose is its high non-specific binding of proteins and otherbio-molecules. Present test strips however often handle a surplus ofsample, reducing the influence of this binding. It is however desirableto minimise the sample volume, in line with the tendency to miniaturizethe entire test, including minimising the amounts of reagents, withoutcompromising accuracy and reliability.

PRIOR ART

EP 1 371 984 discloses a chromatographic assay device and method fordetecting the presence of an analyte in a sample of whole blood,utilizing a red blood cell separating agent to aggregate red blood cellsand permit plasma or serum to flow by capillary action. The carriermaterial is exemplified as a paper (fibrous) material, or a membrane ofcellulose, fibreglass, cloth, both naturally occurring and synthetic, aswell as porous gels.

Although frequently used and well known in the art, the above carriermaterials are associated with many drawbacks. The structure of thematerials will always vary between different batches, and also withinthe material, due to the random distribution of the fibres, e.g. in afibrous material, or cavities, e.g. in a gel-like material. Similarly,the chemical properties of the material, e.g. the distribution ofchemicals added to the material, will inevitable vary for the samereasons as above.

WO 03/103835 discloses micro fluidic systems comprising a substrate,and, provided on said substrate, at least one flow path interconnectingwith functional means in which liquid samples can be subjected todifferent desired procedures, said flow path comprising a plurality ofvertical projections or so called micro posts protruding form saidsubstrate.

SUMMARY OF THE INVENTION

The present invention makes available a device for the separation of acomponent in a liquid sample prior to the detection of an analyte insaid sample, said device having a substrate comprising a zone forreceiving the sample, a reaction zone, and optionally a transport orincubation zone connecting the receiving and reaction zone,respectively, forming a flow path on a substrate, wherein said substrateis a non-porous substrate, and at least part of said flow path consistsof areas of projections substantially vertical to said surface, andhaving a height (H), diameter (D) and reciprocal spacing (t1, t2) such,that lateral capillary flow of said liquid sample in said zone isachieved, and where means for separation are provided within or adjacentto the zone for receiving the sample.

The invention also makes available a method for use in the detection ofan analyte in a liquid sample, said detection taking place in a processon a substrate, where at least a subset of said sample is transportedthrough capillary action from a receiving zone where said sample isadded, to a zone where a reaction/detection takes place, said distancebeing defined as a flow path, wherein said substrate is a non-poroussubstrate, at least part of said flow path consists of areas ofprojections substantially vertical to said surface, and having a height(H), diameter (D) and reciprocal spacing (t1,t2) such, that lateralcapillary flow of said liquid sample is achieved, and that separation ofunwanted components is performed without interruption of said capillaryflow.

The invention also encompasses embodiments of said device and method, asset forth in the description and claims, hereby incorporated in theirentirety by reference.

SHORT DESCRIPTION OF THE DRAWINGS

The invention will be described in closer detail in the followingdescription, examples, and attached drawings, in which

FIG. 1 a shows schematically an embodiment of the present invention,where a drop of sample is added to a substrate 10 having thereon amultitude of projections 12 substantially vertical to said surface. Anarrow 14 indicates the direction of flow.

FIG. 1 b is a partial view of the embodiment shown in FIG. 1 a, showingone embodiment of said vertical projections, said projections having aheight (H), diameter (D) and reciprocal spacing (t1, t2) such, thatlateral capillary flow of said liquid sample in said zone is achieved.

FIG. 2 shows schematically another embodiment of the present invention,where a drop of sample is added to a substrate 20 to an area A1substantially without projections, bordering to a second area A2, havingsuch projections 22. Over the area A2 the height (H), diameter (D) andreciprocal spacing (t1, t2) of the projections is varied so, that agradual retaining or filtration effect is achieved, whereas theneighbouring area A3, acts as a transport and/or reaction zone.

FIG. 3 a shows schematically an embodiment of the present invention,where the sample-receiving zone or area A1 is lowered in relation to theremaining surface of the substrate 30, forming a threshold 34 betweenthe sample-receiving zone, and the remaining lateral flow path. On thethreshold 34 a second area A2 is provided, having first projections 36supporting lateral flow but acting as a filter before the third andconsecutive areas A3 having second projections 38. A1 thus acts as abasin for particulate substances found in the lateral flow, butprevented from passing the threshold by the projections 36. In thisembodiment, the second area A2 is slightly higher than both thesurrounding areas A1 and A3, and the projections on A2 have a height(H), diameter (D) and reciprocal spacing (t1, t2) different from that ofthe projections on A3 and possible consecutive areas (not shown).

FIG. 3 b is a partial view of the embodiment shown in FIG. 3 a,illustrating the dimensions of the first projections 36.

FIG. 4 a shows schematically an embodiment of the present invention,where the lowered sample-receiving zone or area A1 provided in asubstrate 40 has lateral grooves or ridges 42 leading to a threshold 44between the sample-receiving zone and the remaining lateral flow path.

FIG. 4 b shows a partial view of the embodiment shown in FIG. 4 a,illustrating how, on the upper surface of the threshold 44, projections46 are provided, different in size and/or configuration from theprojections 48 in the remaining lateral flow path. Where the ridges 42meet the proximal end of the threshold, elevated portions 43 areprovided, engaging with and bridging the proximal edge of the threshold.Similarly, at the distal edge of the threshold, L-shaped elements formelevated portions, or “claws” 47, engaging and bridging the distal edgeof the threshold. Said details 43 and 47 merely exemplify adaptations ofthe surfaces, improving the capillary action. In the separation of redblood cells, said projections 46 preferably have a height of at leastabout 10 μm and are positioned at a distance of about 3 μm from eachother. The present partial view shows how the projections 46 arepositioned single file, in a meandering pattern on the threshold 44.Several alternative patterns or arrangements are contemplated.

FIG. 5 a schematically shows an alternative embodiment of the above,where on a substrate 50, two compartments are separated by a threshold,wherein the first compartment has longitudinal grooves and ridgesleading up to said threshold and engaging with the same. Said secondcompartment preferably comprises the remaining functions normallyassociated with an assay, reaction and detection zones, etc.

FIG. 5 b shows a partial view of the above embodiment, indicating analternative embodiment where, on the surface of the threshold 54, thevertical projections are arranged in substantially parallel lines ofprojections, here denoted 56′, 56″, and 56′″. These may be the same ordifferent, the difference being the shape, height, width, and distancebetween the projections. Again, where the ridges 52 meet the proximalend of the threshold 54, elevated portions 53 are provided, engagingwith and bridging the proximal edge of the threshold. Similarly, at thedistal edge of the threshold, L-shaped elements form elevated portions,or “claws” 57, engaging and bridging the distal edge of the threshold.Said details 53 and 57 merely exemplify adaptations of the surfaces,improving the capillary action.

FIGS. 6 a, b and c show schematically how magnetic means for separationof unwanted components of the sample can be arranged. While the magnet Mis shown as positioned outside and separated from the substrate 60 inboth FIGS. 6 a and 6 b, and in or on the substrate in FIG. 6 c, theseare only illustrations, and the invention encompasses embodiments wherethe magnet is integrated in the substrate, arranged adjacent to or at adistance from the substrate, as long as the magnetic force is sufficientto aid in the separation in question.

DESCRIPTION Definitions

Before the present device and method is described, it is to beunderstood that this invention is not limited to the particularconfigurations, method steps, and materials disclosed herein as suchconfigurations, steps and materials may vary somewhat. It is also to beunderstood that the terminology employed herein is used for the purposeof describing particular embodiments only and is not intended to belimiting since the scope of the present invention will be limited onlyby the appended claims and equivalents thereof.

It must also be noted that, as used in this specification and theappended claims, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to a reaction mixture containing “an antibody”includes a mixture of two or more antibodies.

The term “about” when used in the context of numeric values denotes aninterval of accuracy, familiar and acceptable to a person skilled in theart. Said interval can be ±10% or preferably ±5%.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outherein.

The term “sample” here means a volume of a liquid, solution orsuspension, intended to be subjected to qualitative or quantitativedetermination of any of its properties, such as the presence or absenceof a component, the concentration of a component, etc. The sample may bea sample taken from an organism, such as a mammal, preferably a human;or from the biosphere, such as a water sample, or an effluent; or froman technical, chemical or biological process, such as a process ofmanufacturing, e.g. the production of medicaments, food, and feed,including cell culture and fermentation, or the purification of drinkingwater or the treatment of waste effluents. The sample may be subjectedto qualitative or quantitative determination as such, or after suitablepre-treatment, such as homogenisation, sonication, filtering,sedimentation, centrifugation, heat-treatment etc.

Typical samples in the context of the present invention are body fluidssuch as blood, plasma, serum, lymph, urine, saliva, semen, gastricfluid, sputum, tear fluid etc.; environmental fluids such as surfacewater, ground water, sludge etc.; and process fluids such as milk, whey,broth, nutrient solutions, cell culture medium, etc. The presentinvention is applicable to all samples, but preferably to samples ofbody fluids, and most preferably to whole blood samples.

The determination based on lateral flow of a sample and the interactionof components present in the sample with reagents present in the deviceand detection of such interaction, either qualitatively orquantitatively, may be for any purpose, such as diagnostic,environmental, quality control, regulatory, forensic or researchpurposes. Such tests are often referred to as chromatography assays, orlateral flow assays, as in e.g. immunochromatography assays.

Examples of diagnostic determinations include, but are not limited to,the determination of analytes, also called markers, specific fordifferent disorders, e.g. chronic metabolic disorders, such as bloodglucose, blood ketones, urine glucose (diabetes), blood cholesterol(atherosclerosis, obesitas, etc); markers of other specific diseases,e.g. acute diseases, such as coronary infarct markers (e.g. troponin-T),markers of thyroid function (e.g. determination of thyroid stimulatinghormone (TSH)), markers of viral infections (the use of lateral flowimmunoassays for the detection of specific viral antibodies); etc.

Another important field of diagnostic determinations relate to pregnancyand fertility, e.g. pregnancy tests (determination of i.a. humanchorionic gonadotropin (hCG)), ovulation tests (determination of i.a.luteneizing hormone (LH)), fertility tests (determination of i.a.follicle-stimulating hormone (FSH)) etc.

Yet another important field is that of drug tests, for easy and rapiddetection of drugs and drug metabolites indicating drug abuse; such asthe determination of specific drugs and drug metabolites (e.g. THC) inurine samples etc.

The term “analyte” is used as a synonym of the term “marker” andintended to encompass any substance that is measured quantitatively orqualitatively.

The terms “zone”, “area” and “site” are used in the context of thisdescription, examples and claims to define parts of the flow path on asubstrate, either in prior art devices or in a device according to theinvention.

The term “reaction” is used to define any reaction, which takes placebetween components of a sample and at least one reagent or reagents onor in said substrate, or between two or more components present in saidsample. The term “reaction” is in particular used to define a reaction,taking place between an analyte and a reagent as part of the qualitativeor quantitative determination of said analyte.

The term “substrate” here means the carrier or matrix to which a sampleis added, and on or in which the determination is performed, or wherethe reaction between analyte and reagent takes place.

The term “chemical functionality” comprises any chemical compound ormoiety necessary for conducting or facilitating the assay. One group ofchemical compounds, with particular relevance in the present invention,are compounds or components exhibiting specific affinity to, orcapability of binding or interacting with, one or more components in thesample. Red blood cell separating agents constitute an illustrativeexample. Such agents may be any substance capable of aggregating orbinding red blood cells.

Preferred agents are positively charged materials such as polycations,including e.g., poly-L-lysine hydrobromide; poly(dimethyl diallylammonium) chloride (Merquat™-100, Merquat™ 280, Merquat™ 550);poly-L-arginine hydrochloride; poly-L-histidine; poly(4-vinylpyridine),poly(4-vinylpyridine) hydrochloride; poly(4-vinylpyridine)cross-linked,methylchloride quaternary salt; poly(4-vinylpyridine-co-styrene);poly(4-vinylpyridinium poly(hydrogen fluoride));poly(4-vinylpyridinium-P-toluene sulfonate);poly(4-vinylpyridinium-tribromide);poly(4-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate); polyvinylpyrrolidone, cross-linked; poly vinylpyrrolidone,poly(melamine-co-formaldehyde); partially methylated; hexadimethrinebromide; poly(Glu, Lys) 1:4 hydrobromide; poly(Lys, Ala) 3:1hydrobromide; poly(Lys, Ala) 2:1 hydrobromide; poly-L-lysinesuccinylated; poly(Lys, Ala) 1:1 hydrobromide; and poly(Lys, Trp) 1:4hydrobromide. The most preferred polycation is poly (dimethyl diallylammonium) chloride (Merquat™-100).

The red blood cell separating agent may be used in any suitable amount,which functions to separate the red blood cells from the rest of thesample. Preferably, the red blood cell separating agent may be presentin a concentration of from about 0.04% to about 1.3% (weight pervolume), with from about 0.13% to about 0.33% (weight per volume) beingmore preferred, and about 0.20% to about 0.33% (weight per volume) beingmost preferred.

A positive charge on the red blood cell separating agent has a tendencyto aggregate any negatively charged agent present on the strip. Forexample, a labeled substance or conjugate bound to the chromatographycarrier may also be aggregated by the red blood cell separating agentinterfering with binding of the analyte to the conjugate or, in acompetitive assay, the binding of the labeled substance and the analyteof interest to the trapping substance at the detection site or aconjugate. Ultimately, the sensitivity and accuracy of the immunoassaysystem may be compromised.

Accordingly, when the blood cell separating agent is a positivelycharged material, the present invention preferably employs aneutralization agent. The neutralization agent is capable ofneutralizing the positive charge of the red blood cell separating agent,thereby eliminating or at least minimizing any interference to the assaysystem caused by the red blood cell separating agent. Preferably, theneutralization agent is diffusively bound to the chromatographiccarrier. The neutralizing agent may be diffusively bound at any locationon the chromatographic carrier where it will function to neutralize ared blood cell separating agent, but is preferably located downstream ofthe red blood cell separating agent and upstream of the detection site,and more preferably is located at the same place on the chromatographiccarrier as the diffusively bound labeled substance.

The neutralizing agent may be any polyanion capable of neutralizing thepositive charge of the red blood cell separating agent. Preferredpolyanions include poly(acrylic acid), poly(acrylic acid, Na salt),poly(methyl methacrylic acid), poly(Na-4-styrene sulfonate), poly(vinylsulfonic acid), poly-L-aspartic acid, and carboxymethyl cellulose, withdextran sulfate being the most preferred.

The neutralization agent may be present in any amount which functions toneutralize the positive charge of the red blood cell separating agent.Generally, the concentration of the neutralization agent is dependentupon the concentration of the red blood cell separating agent beingused. Preferably, the neutralizing agent is present in a concentrationof from about 0.33% to about 20% (weight per volume), with about 0.34%to about 10% (weight per volume) being more preferred and 0.34% to 10%(weight per volume) being most preferred.

The term “biological functionality” comprises all biologicalinteractions between a component in a sample and a reagent on or in thesubstrate, such as catalysis, binding, internalisation, activation, orother biospecific interaction. Suitable reagents include, but are notlimited to, antibodies, antibody fragments and derivates, single chainantibodies, lectines, DNA, aptamers, etc., including other polymers ormolecules with binding capacity. Such reagents can be identified by aperson skilled in the art, following the choice of the component to beseparated, using standard experimentation, e.g. screening methods andchemical libraries.

The term “physical functionality” here comprises functionalitiesinvolved in reactions and interactions other than those that are mainlychemical or biological. Examples include diameter, height, shape, crosssection, surface topography and surface patterns, the number ofprojections per unit area, wetting behavior of the surface of saidprojections, or a combination thereof, and/or other functionalitiesinfluencing the flow, retention, adhesion or rejection of components ofthe sample.

The distinctions between chemical, biological and physical interactionsare not always clear, and it is possible that an interaction, such as aninteraction between a component in a sample and a reagent on thesubstrate, involves both chemical, biological and physical elements. Theterms “hydrophilic” and “hydrophobic”, as in hydrophilic or hydrophobiccompounds, hydrophilic or hydrophobic interactions etc., have themeaning generally understood by a person skilled in the art, andcorresponding to that used in generally recognised textbooks.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention makes available a device and method for theseparation of a component in a liquid sample during or prior to thedetection of an analyte in said sample, wherein a sample is added to areceiving zone on a substrate, said substrate optionally furthercomprising a reaction zone, a transport or incubation zone connectingthe receiving and reaction zone, respectively, forming a flow path on asubstrate, wherein said substrate is a non-porous substrate, and atleast part of said flow path consists of areas of projectionssubstantially vertical to said surface, and having a height (H),diameter (D) and reciprocal spacing (t1, t2) such, that lateralcapillary flow of said liquid sample in said zone is achieved, and wheremeans for separation are provided adjacent to the zone for receiving thesample. Said means for separation are chosen among filter means,optionally enhanced by affinity binding and/or aggregation; magneticmeans, also optionally enhanced by affinity binding and/or aggregation;and acoustic means.

The device and method are further defined in the attached claims,incorporated herein by reference.

The device according to the present invention is built on a plasticsubstrate, preferably thermoplastic, or a substrate having a plasticupper layer. This can in turn be coated or derivatised, e.g. usingtechniques such as sputtering, vapour deposition and the like, and givena coating of silicon, a metal or other. The present invention can alsobe made of silicon or glass substrates. According to a preferredembodiment the substrate is given a hydrophilic treatment or coating,e.g. by subjecting the substrate to an oxidative treatment, such as e.g.gas plasma treatment, coating with a hydrophilic substance such assilicon oxide, hydrophilic polymers such as dextran, polyethyleneglycol, heparin and derivatives thereof, detergents, biologic substancessuch as polymers, etc.

According to one embodiment of the present invention, said means forseparation comprise a filter or filtering zone, located on saidsubstrate in said flow path, said filter comprising projectionssubstantially vertical to said surface, and having a height (H),diameter (D) and reciprocal spacing (t1, t2) such, that lateralcapillary flow of said liquid sample in said zones is achieved, whilethe components to be separated are substantially prevented from passingsaid filter.

According to another embodiment, said filtering zone is providedadjacent to the zone for receiving the sample, said filtering zonehaving projections substantially vertical to its surface, having aheight (H), diameter (D) and reciprocal spacing (t1, t2) forming agradient with regard to the diameter (D) and/or reciprocal spacing (t1,t2) such that components of the sample are gradually retained.

According to a preferred embodiment of the invention, said receivingzone further contains means enhancing the separation capability of saidmeans for separation. Said means are preferably predispensed in thereceiving zone. Preferably, said means are compounds capable of formingaggregates of said component to be separated. Such compounds or meansare chosen among those defined as having a chemical, biological and/orphysical functionality as defined above.

According to one embodiment, said means are beads, derivatised with orcarrying on their surface compounds capable of forming aggregates ofsaid component to be separated.

Suitable beads are available from different suppliers, eitherderivatised or “naked”. One example is the Dynabead® product line,available from Dynal Biotech A/S, Oslo, Norway.

According to another embodiment, said reciprocal spacing (t1, t2) is inthe interval of 1-100 μm, and preferably said spacing varies within saidmeans for separation, forming a gradient in the direction of the flow.According to a particular embodiment, suitable for the removal of redblood cells, said spacing varies from about 7 to about 1 μm.

According to another embodiment, said receiving zone forms a basincapable of containing the part of the sample separated by the means forseparation.

A general embodiment is illustrated in FIGS. 1 a and 1 b, showing a partof a device where the surface of a substrate is covered by projectionssubstantially vertical to said surface, and having a height (H),diameter (D) and reciprocal spacing (t1, t2) such, that lateralcapillary flow is achieved.

Another embodiment is illustrated in FIG. 2, showing a part of a devicecomprising a substrate 20 with vertical projections 22 on its surface,where the sample-receiving area A1 does not have such projections on itssurface, but where the adjacent area A2 has projections forming agradient, and the subsequent area A3 functioning as a zone or zones forincubation, transport, reaction, and/or detection.

According to an embodiment, means for separation are predispensed in thesample receiving zone, preferably in a form that allows their dispersionor solution upon addition of the sample. Said means can be chemical orbiochemical compounds deposited, e.g. lyophilised on the substrate.Suitable methods and auxiliary agents can be used, as well known to askilled person familiar with techniques for deposition reagents onsurfaces.

Further, according to another embodiment of the invention, theseparation is based on affinity reactions, and said projections or atleast a sub-set thereof are provided with a chemical, biologic orphysical functionality, as defined above. The projections may havechemically reactive groups on their surface. The projections may alsohave substances with biological affinity on to their surface.

According to a related embodiment, the projections carry structures orgroups having a chemical, biologic or physical functionality, as definedabove.

Particularly suitable are compounds or groups binding to the particlesin question, e.g. the red blood cells. Sugar binding molecules are oneexample, and in particular lectines, which have been shown to be capableof agglutinating the red blood cells. Further examples includepreferably multivalent compounds or constructs, i.e. compounds orconstructs presenting two (bivalent) or more binding groups(multivalent). Antibodies and similar reagents are examples of bivalentbinders.

Multivalent constructs can also be made on the base of commerciallyavailable particles, e.g. so called beads, acting as the core for one ormore binding groups, or based on a neutral compound, e.g. albumin, towhich two or more binding groups are attached. Suitable beads areavailable from different suppliers, either derivatised or “naked”. Oneexample is the Dynabead® product line, available from Dynal Biotech A/S,Oslo, Norway. Techniques for the adaptation or derivatisation of beads,and for the coupling of functional groups to e.g. albumin are known inthe art. Suitable binders can be identified without inventive effort bya person skilled in the art, following the choice of the component to beseparated. Possible derivatisation of commercial beads lies also withthe skills of a person familiar with the relevant art.

According to yet another embodiment, the projections have a physicalproperty selected from the projection diameter (D), height (H),reciprocal spacing (t1, t2), shape, cross section, surface coating, thenumber of projections per unit area, wetting behavior of the surface ofsaid projections, or a combination thereof, according to the desired enduse of the substrate.

According to another embodiment, particles are provided chemically orphysically bound to the substrate, or mechanically trapped within aregion comprising a plurality of projections. Said particles are chosenamong commercially available particles, so called beads, and may have acore of glass, metal or polymer, or a combination of these, and theyoptionally carry on their surface one or more chemical, biological orphysical functionality, as defined above. Preferably, said beads have,bound to their surface, agents with specific affinity to the componentto be separated. More preferably, said beads have a magnetic core. Suchbeads, either “naked” or derivatised, are commercially available, forexample from Dynal Biotech A/S, supra. Possible derivatisation of suchbeads can be performed by a skilled person, using known or slightlymodified protocols, or according to the manufacturer's instructions.

According to a preferred embodiment of the invention, the inventivedevice either comprises a magnet, or is adapted to be used inconjunction with a magnet. Preferably said magnet is a permanent magnetor an electromagnet. When said magnet is incorporated into the device,it is preferably an electromagnet, which can be activated using a signalor impulse coming from auxiliary equipment outside the device itself.

According to another embodiment of the device, said receiving zone formsa basin capable of containing the part of the sample separated from theflow by the means for separation. In this embodiment, said magnet ispreferably positioned in the vicinity of said basin, and most preferablybelow or upstream of the receiving zone.

According to one embodiment, magnetic or paramagnetic beads are used,said beads being derivatised with groups having affinity for theparticles to be separated from the flow, or affinity for groups on theseparticles. Suitable magnetic beads are available from differentsuppliers, either derivatised or “naked”. One example is the Dynabead®product line, available from Dynal Biotech A/S, Oslo, Norway. Anotherexample are the functional magnetic beads from Bioclone Inc., San Diego,Calif., USA. These are uniform superparamagnetic 1 μm or 5 μm diameterbeads with different surface groups such as amine, carboxy, aldehyde,epoxy, IDA, hydrazide, NADPA etc. As noted above, possibly necessaryderivatisation of such beads can be performed by a skilled person, usingknown or slightly modified protocols, or according to the manufacturer'sinstructions.

The magnetic beads are brought in contact with the sample, and allowedto attach to the particles to be separated. Magnetic force is thenapplied to the sample, preferably in or near the proximal end of thelateral flow path, in order to retain the beads with the particles boundthereto at an early stage. Magnetic force can be applied externally,e.g. by bringing a magnet in close association with the flow path,either below, above or at either side of the flow path, preferably belowor at the side of the flow path. Magnetic force can also be applied byincorporating a magnet or magnetic particles in the substrate, at apoint below or at the side of the lateral flow path. These embodimentsare schematically illustrated in FIGS. 6 a, b and c.

In FIG. 6 a, an embodiment is shown, where a flow path is provided on asubstrate 60, the direction of flow being indicated by an arrow A fromleft to right. A sample is added to a receiving zone 62, in which zonemagnetic particles, derivatised to exhibit affinity to the components tobe separated, are predispensed. Upon addition of the sample, theparticles mix with the sample, and the mixture travels laterally in thedirection A through capillary action. In a separation zone indicated as64, the particles are subjected to a magnetic force, drawing theparticles away from the main flow, as indicated by the arrow B. Themagnetic force can originate from a permanent magnet or anelectromagnet, here denoted M, either incorporated into the substrate 60(not shown) or positioned in the vicinity of said substrate. The sampleflow, depleted of the component removed by the magnetic action,continues into further reaction and detection zones, here indicated as66.

Another embodiment is illustrated in FIG. 6 b, where on a substrate 60,the sample is added to a sample receiving zone 63, containingpredispensed magnetic particles, derivatised to exhibit affinity to thecomponents to be separated. Said zone 63 is preferably slightly lowerthan the remaining flow path, but can also be level with the same. Thesample receiving zone can contain substantially vertical projections orlack such projections. In the vicinity of the receiving zone 63, andpreferably below said zone, a magnet M is positioned. Preferably saidmagnet, when a permanent magnet, is brought into the vicinity of thereceiving zone a certain time after addition of the sample, sufficientfor the reaction between the sample and the magnetic particles to takeplace. When said magnet M is an electromagnet, it is activated only acertain time after addition of the sample, sufficient for the reactionbetween the sample and the magnetic particles to take place. When thepermanent magnet is in place or the electromagnet activated, componentsbound to derivatised particles will be prevented from leaving the zone63, and the remaining sample, depleted with respect to unwantedcomponents, will be drawn by capillary action along the flow pathtowards subsequent reaction, incubation and detection zones (not shown).

Yet another embodiment is illustrated in FIG. 6 c, where a magnet M ispositioned along the flow path on a substrate 60. Sample is added to asample receiving zone 62 containing predispensed magnetic particles,derivatised to exhibit affinity to the components to be separated. Thesample and said particles will be drawn in the direction of the arrow Aby capillary action, and when reaching the magnet M positioned alongsidethe flow path, the magnetic particles will deviate from the flow, asindicated by the arrows B. Said magnet is either a permanent magnet, oran electromagnet, activated a certain time after addition of the sample,sufficient for the reaction between the sample and the magneticparticles to take place.

The above embodiments may also be combined, e.g. in an embodiment wherea depressed area borders to a raised area, or an area in level with theremaining surface of the substrate, of raised above said surface, andwhere the projections are placed on the border between said depressedand raised areas, the distance between said posts, as well as theirwidth, height and shape, being chosen so, that when a sample of wholeblood is added to said depression, the red blood cells are preventedfrom leaving the depression, whereas the plasma or serum will flowthrough said area having vertical projections.

The device according to the invention is advantageously used inanalytical applications where the liquid sample contains particulatematter, such as cells, tissue debris, organic or inorganic matter, othercontamination etc, which is desired to separate from the bulk of thesample. One important application is when the liquid sample is wholeblood and in such cases, the lateral capillary flow involves theseparation of red blood cells from plasma without significant rupture ofsaid cells. According to one embodiment, such separation in general, andin particular the gentle separation of red blood cells, is achieved in agradient of projections wherein the spacing (t1, t2) decreases fromabout 7 μm to about 1 μm over the length of said filtering zone.

According to one embodiment said receiving zone forms a basin forcomponents separated from the lateral flow, e.g. particulate matter orcells prevented from passing between the projections, or entering thatspace only to a limited degree. Said basin is formed in that the flow ofliquid is restricted, i.e. prevented or delayed, in one or preferablymore directions. When the basin has a substantially square orrectangular shape, the flow of liquid is restricted, preferablyprevented, in three directions, the forth direction being the one wherethe liquid is forced to pass over or through a filtering zone. If thebasin is substantially round or elliptic, the flow of liquid isrestricted, preferably prevented over at least 180 degrees of itscircumference, preferably 210 degrees and most preferably about 270degrees of its circumference. The restriction or prevention of liquidflow may consist in walls or raised portion surrounding the basin. Therestriction of flow may also consist in the basin being at leastpartially surrounded by material that does not support lateral flow,e.g. material that does not support capillary transport due to theconfiguration, density etc of the material, non-porous material,material impregnated with agents repelling the liquid, e.g. stronglyhydrophobic material etc.

According to another embodiment, the basin is lowered in relation to theplane of the lateral flow. Preferably said filtration zone is arrangedas the border leading to the remaining lateral flow path. Morepreferably, the filtration zone is arranged on a raised portion, forminga threshold between said basin and the lateral flow path.

According to one particular embodiment for the separation of componentsfrom a liquid sample, e.g. red blood cells from plasma, said device hasan area, optionally a depressed area, surrounded on at least part of itscircumference by projections, the distance (t1, t2) between saidprojections, as well as their diameter (D), height (H) and shape, beingchosen so, that when a sample of whole blood is added to said area, thered blood cells are prevented from leaving said area by the projectionsdown stream of said depression, whereas the plasma will flow through orbetween said projections.

According to an embodiment of the invention, the particulate matterpresent in the sample is particulate matter, which is capable oftravelling with the lateral flow. In applications where said liquidsample is whole blood, it is important that said lateral capillary flowinvolves the transportation (when applicable) and an optional subsequentseparation of red blood cells without significant rupture of said cells.This is achieved by the present invention through the control of one ormore of the parameters of the projections, such as the height (H),diameter (D) and reciprocal spacing (t1, t2), as well as the chemical orbiochemical derivatisation of the projections.

The spacing (t1, t2) between said projections can be varied depending onthe intended use and the properties of the liquid sample, as well as theproperties of components to be separated or transported, and preferablyis in the interval of 1 to 100 μm, more preferably in the interval of 1to 50 μm. The distance between said projections can be chosen by askilled person, considering which sample the device is intended for, theproperties of said sample, and the properties of the components that areto be separated.

In a particularly preferred embodiment, the separation is achieved bothby the action of gravity and a filtering action. In this embodiment,schematically illustrated in FIGS. 3 a, 3 b, 4 a, 4 b, 5 a, 5 b, and 6b, the provision of a first volume or basin help to retain part of theunwanted particles, as they are less capable of passing over thethreshold 34, 44, and 54, and therefore sediment in the basin. Further,the provision of vertical projections 36, 46, and 56′, 56″, 56′″function as a filter, separating the particles from the liquid. Inapplications where the function of the filter is to separate red bloodcells from plasma, these vertical projections preferably have a heightof about 10 μm and a mutual distance of about 3 μm. For the separationof other particles, different dimensions are contemplated. Also, whenthe separation is aided by other means, such as magnetic and/orchemically derivatised beads, multivalent chemical compounds etc.,different dimensions may be used.

In the embodiment shown as FIGS. 3 a and 3 b, the vertical projections36 cover substantially the entire surface of the threshold 34. It isunderstood that the projections can be arranged in other configurations,as long as they form a continuous filter barrier across the flow path.FIGS. 4 a and 4 b show an embodiment where the projections 46 arearranged single-file in a meandering fashion over the surface of thethreshold 44. Further, in this figure, means 43 and 47 are shown, where43 indicates extensions of the ridges 42 engaging the proximal edge ofthe surface 44, and 47 indicates L-shaped members engaging the distaledge of the surface 44, and leading into the remaining flow path. Themeans 43 and 47 enhance the liquid flow over the threshold.

According to yet another embodiment, ultrasonic standing waves are usedto separate particle present in a liquid flow, in a flow path on or in adevice according to the invention, based on the acoustic response of theparticles. Particles suspended in a liquid medium are known to collectin the nodes of standing ultrasonic waves, and this has been usedseparation purposes in the past.

For example Riera-Franco de Sarabia et al., (Application of high-powerultrasound to enhance fluid/solid particle separation processes,Ultrasonics. 2000 March; 38(1-8): 642-6), present possibilities to useultrasonic energy to assist conventional separation techniques.Ultrasonic separation has also been applied in surgery, e.g. for theseparation of lipid particles in blood during major surgery,significantly reducing embolic load to the brain after cardiac surgery.See e.g. Jonsson et al. (Particle separation using ultrasound canradically reduce embolic load to brain after cardiac surgery, Ann ThoracSurg. 2004 November; 78(5): 1572-7; discussion 1577-8. According toJonsson et al., the mean separation rates for lipid particles were81.9%+/−7.6% and for erythrocytes 79.8%+/−9.9%, and both were related tothe hematocrit level of the incoming blood sample. Importantly, theprocedure was atraumatic and did not cause hemolysis. The authorsconclude that particle separation by means of an acoustic standing-wavetechnique can be used for atraumatic and effective removal of lipidparticles from blood, with the possible clinical implication of reducingneurocognitive complications after cardiopulmonary bypass. The authorsof the cited article are also the inventors behind the patent SE0100819-2 concerning a device for the separation of suspended particlesfrom a fluid using ultrasound and method of such separation.

According to the present invention, ultrasonic standing waves arecreated in the lateral flow, preventing the unwanted particles fromadvancing, and facilitating the flow of particle depleted sample furtherdown the flow path. One example is the immobilization of red blood cellsusing standing waves, where blood cell depleted plasma will be drawnalong the flow path by capillary action.

The standing wave is created by arranging either two transducers of thesame frequency facing each other through the liquid flow path, or by areflector facing a single transducer, or by several transducer-reflectorpairs. The transducers or the reflector and transducer can be arrangedabove and below the flow path, to either side of the flow path, orotherwise making sure that the nodes of the standing waves are formedwithin the flow path.

Thus, according to one embodiment, said means for subjecting the sampleto ultrasonic standing waves comprise at least two ultrasonic energysources arranged to establish a pattern of nodes within the flow path byinterference between their outputs defining a standing wave.

According to an alternative embodiment, said means for subjecting thesample to ultrasonic standing waves comprise at least one ultrasonicenergy source and a reflector, or two ultrasonic energy sources withcorresponding reflectors, arranged to establish a pattern of nodeswithin the flow path by interference between their outputs defining astanding wave.

Said transducer(-s) and reflector may be incorporated in the deviceaccording to the invention, or provided in an auxiliary device, e.g.incorporated in a device used for reading the result. More informationon the creation of standing waves can be found in general textbooks onultrasonics, e.g. “Fundamentals of Ultrasonics, by J. Blitz,Butterworths, 1967. Ultrasonic filters are also disclosed in patents,see e.g. UK 2098498, inventor: R. Sayles, and EP 147 032, inventor: C.J. Schram.

The present invention also makes available a device suitable for use inor together with a device for detection of an analyte in a liquidsample, wherein said device has projections substantially vertical toits surface, said projections having a height (H), diameter (D) andreciprocal spacing (t1, t2) such, that said device is capable ofseparating components of said liquid sample while achieving a lateralflow of said liquid sample. This device may have one or more of theproperties and functionalities described above, depending on itsintended use.

This device may be used separately, in association with, or integratedin a device for the analysis of a liquid sample. This device mayfunction as a pre-treatment step in or before a conventional analysis.

The present invention also makes available methods for performing anassay on a liquid sample, said sample being applied to a substratehaving a zone for receiving the sample, which is in fluid connectionwith a reaction zone, and optionally a transport or incubation zoneconnecting the receiving and reaction zone, respectively, wherein saidsubstrate is a non-porous substrate, and said receiving zone, anoptional reaction zone, transport or incubation zone, consist of areasof projections substantially vertical to said surface, and having aheight (H), diameter (D) and reciprocal spacing (t1,t2) such, thatlateral capillary flow of said liquid sample in said zone is achieved.

According to one embodiment of these methods, a filtering step isperformed following the addition of the sample, said filtering effectedin a filtering zone by projections substantially vertical to the surfaceof said substrate, the projections having a height (H), diameter (D) andreciprocal spacing (t1, t2) forming a gradient with regard to thediameter (D) and/or reciprocal spacing (t1, t2) such that components ofthe sample are gradually retained.

Said reciprocal spacing (t1, t2) is preferably in the interval of about1 to about 100 μm, more preferably from about 7 to about 1 μm.

Alternatively, said separation is achieved using filtering means havingprojections substantially vertical to the surface of said substrate, andhaving a height (H), diameter (D) and reciprocal spacing (t1, t2) such,that the compound to be separated from the sample is substantiallyprevented from leaving the receiving zone.

In the method according to the invention, means enhancing the separationcapability of said means for separation are preferably provided in saidreceiving zone. Said means are preferably compounds capable of formingaggregates of said component to be separated.

According to one embodiment, said means are beads, derivatised with orcarrying on their surface compounds capable of forming aggregates ofsaid component to be separated.

According to another embodiment, the part of the sample separated by themeans for separation is contained in a basin, formed by said receivingzone.

According to a preferred embodiment of the invention, said separation isenhanced by means having specific affinity to the component to beseparated and said means are provided in the flow path. Preferably saidmeans are projections substantially vertical to the surface of saidsubstrate, and having a height (H), diameter (D) and reciprocal spacing(t1, t2) such, that capillary flow of the sample is possible, and saidprojections are provided with, bound to their surface, agents withspecific affinity to the component to be separated. Alternatively, saidmeans are beads having, bound to their surface, agents with specificaffinity to the component to be separated.

Preferably, said beads have a magnetic core. Suitable magnetic beads areavailable from different suppliers, either derivatised or “naked”. Oneexample is the Dynabead® product line, available from Dynal Biotech A/S,Oslo, Norway. Another example are the functional magnetic beads fromBioclone Inc., San Diego, Calif., USA. These are uniformsuperparamagnetic 1 μm or 5 μm diameter beads with different surfacegroups such as amine, carboxy, aldehyde, epoxy, IDA, hydrazide, NADPAetc. A skilled person will be able to, without undue burden, to ordersuitable beads or to modify existing beads. Modification orderivatisation can be performed according to existing protocols,modified protocols, or according to the manufacturer's instructions, bya person skilled in the art.

According to a preferred embodiment, said beads are retained or removedfrom the flow by a magnet arranged in or adjacent to said device. Saidmagnet may be a permanent magnet or an electromagnet.

According to an embodiment of the invention, the part of the sampleseparated from the flow by the means for separation is contained in abasin, formed by said receiving zone. In this embodiment, when using amagnet, said magnet is positioned in the vicinity of said basin,preferably below or behind said receiving zone, in relation to thedirection of the flow.

According to another embodiment of the present invention, saidseparation is enhanced by subjecting the sample to ultrasonic standingwaves. Preferably the sample is subjected to ultrasonic standing wavesby at least two ultrasonic energy sources arranged to establish apattern of nodes by interference between their outputs defining astanding wave within the flow path.

Alternatively, said sample is subjected to ultrasonic standing waves byat least one ultrasonic energy source and a reflector, or two or moresources and their corresponding reflectors, arranged to establish apattern of nodes by interference between their outputs defining astanding wave within the flow path.

These methods can be used for all applications where components of aliquid sample need to be separated from the bulk of the sample. Themethods are however particularly suitable for applications where saidliquid sample is whole blood and said lateral capillary flow involvesthe separation of red blood cells from plasma without significantrupture of said cells.

The device according to the present invention surprisingly replacesprior art devices where the substrate, and/or one or more of said zoneswere made of a porous material such as nitrocellulose, cellulose,asbestos fibres, glass fibres and the like.

Advantages of the Invention

An advantage of the device according to the invention is the increasedspeed of the determination, as no separation of cellular material isnecessary, or when such separation is desired, rapid separation takesplace.

Another advantage of the device is that, due to the open, regularstructure and the defined properties of the capillary flow zones, theaddition of reagents to these zones or the derivatisation of the surfaceof the projections is greatly simplified.

Yet another advantage of the device is the uniformity of the structurenot only within a single device, but also between all devices produced.This result in significantly increased reliability and repeatability ofthe assays built on the inventive device.

An important advantage of the inventive device is that the degree ofseparation, from none to total, of the blood cells, can be accuratelycontrolled.

The inventive device has many advantages with respect to themanufacturing process. All capillary zones can be made in one step andno assembly of parts is required. Optionally, a cover having at leastone aperture for sample addition and one reading the result of the assaycan be placed over the substrate and the capillary zones.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionwhich is set forth in the claims appended hereto.

The invention claimed is:
 1. A method for use in the detection of ananalyte in a liquid sample, said detection taking place in a process ona substrate, said method comprising the steps of: adding liquid sampleto a receiving zone on a surface of said substrate; transporting atleast a subset of said liquid sample through capillary action from saidreceiving zone where said sample is added, to an optional zone where areaction/detection takes place, said transport by capillary actiondefining a lateral flow path, said substrate being non-porous, at leastpart of said lateral flow path comprising areas of projectionssubstantially vertical to said surface, and having a height (H),diameter (D) and reciprocal spacing (t1, t2) such that lateral capillaryflow of said liquid sample is spontaneously induced without requiring acover to contribute to the induced capillary flow, said substratefurther including a zone opposite said receiving zone exerting capillaryforce and having the capacity to receive liquid sample along said flowpath; and separating unwanted components in said sample withoutinterruption of said capillary flow wherein said separation step isachieved using means for separation comprising a filter that retainssaid unwanted components and wherein a device retaining said substrateis constructed to induce capillary action without the need for a cover.2. The method according to claim 1, including the step of providing saidfilter within the receiving zone.
 3. The method according to claim 1,wherein said means for separation separate specific cells or particulatematter.
 4. The method according to claim 1, including the step ofarranging wherein said projections to drive the flow through said filtersuch that sample fluid is filtered prior to flow.
 5. A method accordingto claim 1, wherein the reciprocal spacing (t1, t2) is about 100 μm toabout 1 μm.
 6. A method according to claim 5, wherein the reciprocalspacing (t1, t2) is about 7 μm to about 1 μm.
 7. The method according toclaim 1, including the step of lowering said sample receiving zonerelative to the remaining surface of said substrate to create athreshold between the sample receiving zone and the remainder of saidlateral flow path.
 8. The method according to claim 7, including thestep of defining two areas of projections in which a first set ofprojections are provided on the threshold and the a second set ofprojections are provided on the remainder of said substrate oppositesaid liquid receiving zone.
 9. The method according to claim 8, whereinsaid first projections are defined by different height, diameters andreciprocal spacings than said second projections.
 10. The methodaccording to claim 9, including the step of lowering said liquidreceiving area as a basin on one side of said threshold opposite saidsecond projections.
 11. The method according to claim 9, wherein thestep of lowering said sample receiving zone includes the step ofproviding a plurality of lateral grooves leading to said thresholdbetween said sample receiving zone and the remainder of said lateralflow path.
 12. The method according to claim 11, including the step ofproviding said first projections on at least a portion of an uppersurface of said threshold.
 13. The method according to claim 11, whereinsaid lateral grooves include ridges, the method including the additionalstep of providing elevated areas bridging the ridges and the uppersurface of said threshold.
 14. The method according to claim 12,including the additional step of providing L-shaped elements on theopposite side of said threshold relative to sample receiving zone, saidL-shaped elements including elevated portions to enable capillary actionto bridge said threshold.
 15. The method according to claim 14, whereinthe first projections provided on said upper surface of said thresholdare defined in parallel lines arranged perpendicular to the lateral flowpath.
 16. The method according to claim 15, wherein at least oneparallel line of said first projections varies in at least one ofheight, width and distance in relation to other said lines of firstprojections.
 17. The method according to claim 14, wherein said firstprojections are provided single file in a meandering pattern on saidupper surface of said threshold.